The present invention relates generally to polycationic buffers for high resolution gel electrophoresis of nucleic acids. More particularly, the present invention relates to the high resolution polyacrylamide gel electrophoresis of nucleic acids comprising oligonucleotides of about 2-70 nucleotides in length using L-histidine buffer.
Polyacrylamide gel electrophoresis, under denaturing conditions, is one of the most widely used methods for separating nucleic acids. The method is used to analyze mixtures of nucleic acids, as well as to purify them on a preparative scale. The term nucleic acids or oligonucleotides, as used herein, includes both single and double-stranded DNA and RNA.
A prevalently used electrophoresis buffer for nucleic acids has been described by Peacock and Dingman. Peacock, A. and Dingman, C. W., Molecular Weight Estimation and Separation of Ribonucleic Acid by Electrophoresis in Agarose-Acrylamide Composite Gels, Biochemistry, 7:668-674 (1968). This buffer, 89 mM Tris-borate, 2 mM EDTA, at pH 8.3, provides adequate separation of DNA or RNA but has disadvantages associated with its use. For example, the buffer's relatively high conductivity makes the entire separation process a lengthy procedure and, in addition, the separation between bands obtained using this Tris-borate buffer is less than the maximal separation theoretically possible. Accordingly, there is a need for a buffer which would give improved separation and resolution of nucleic acids on gel electrophoresis.
Although electrophoretic separation of nucleic acids is not disclosed, the separation of hemoglobins by substituting one of several amino acids, including isoleucine, alanine, valine, histidine, threonine and serine, in the place of glycine, in a discontinuous buffer system utilizing two buffers, the buffer in the upper tank having a higher pH than the buffer in the lower tank, is disclosed in Parkinson, et al., Anal. Biochem., 117:6-11 (1981). The amino acid substitution, at a concentration of 192 mM is stated 1) to alter the running pH of the gel and 2) to change the propagation rate of the moving front, the former modifying the absolute mobility of the proteins and the latter modifying their relative mobilities. The migration distances and the degree of separation is reported to vary from greatest to least as follows: L-isoleucine &gt;DL-alanine &gt;L-alanine &gt;DL-valine &gt;glycine &gt;L-histidine &gt;DL-threonine &gt;L-serine.